Biofabrication of Multicompartment Human Liver Tissues for Chemical Screening

摘要/项目总结 标题：用于化学筛选的多室人肝组织生物制剂 药物性肝损伤（DILI）是临床前和临床药物消耗的主要原因， 毒品，以及从市场上撤回毒品。不幸的是，动物模型并不总是足以 由于药物代谢途径存在显著的种属特异性差异，因此， 人类肝脏的体外模型越来越多地用于评价化合物（药物/化学品） 代谢和毒性。然而，目前人类肝脏的体外模型无法确定其影响 化合物对肝脏的三个主要部分，即肝，血管和胆道，以及如何 对一个隔室的毒性可能影响其他隔室。同样，虽然取得了一些进展， 在开发可植入的肝组织替代物作为基于细胞的疗法，用于患有终末期肝病的患者中， 肝衰竭时，这些组织不含上述具有生理性的肝区室， 相互联系我们的研究表明，原代人肝细胞（PHH）和肝内皮细胞 (LEC)当组织成三维（3D）时，在体外显示高水平的体内样功能4周以上 使用高通量液滴微流体平台产生的细胞外基质（ECM）微凝胶 （所谓的微组织）。这种微组织技术特别适合于控制肝脏的微环境 细胞，并可能保护细胞免受通过3D生物打印诱导的剪切应力。此外，我们有 表明胆管细胞在脱细胞肝细胞外基质（dECM）中表现出发芽行为，但在I型胶原中则不然 或单独的基质胶，并且这种发芽行为可以通过3D生物打印来指导。在这个高风险/高回报的 R21提案，我们将利用这些平台和研究结果来测试新的假设，即3D打印 含有肝微组织和肝dECM的生物材料支架可用于产生肝样功能性 和整合的隔室（血管、肝脏和胆道）。在目标1中，我们将制造和表征3D- 打印包含肝脏微组织和LEC内衬血管通道的结构，而在目标2中，我们将 将胆管细胞纳入生物制造结构并研究控制和检测胆汁的能力 流如果成功，我们的努力将产生第一个可扩展的3D打印人类肝脏组织， 完整的肝脏、血管和胆道隔室，显示稳定的不同肝功能水平， 几周的体外培养最终，我们的3D打印人类肝脏组织可用于研究 化合物对肝脏所有三个隔室的作用及其相互作用，以及用于植入动物体内 作为慢性肝病和急性肝衰竭的潜在细胞治疗模型。

ABSTRACT / PROJECT SUMMARY Title: Biofabrication of Multicompartment Human Liver Tissues for Chemical Screening Drug-induced liver injury (DILI) is a leading cause of preclinical and clinical drug attrition, black box warnings on drugs, and withdrawals of drugs from the marketplace. Unfortunately, animal models do not always suffice to evaluate human DILI due to significant species-specific differences in drug metabolism pathways; therefore, in vitro models of the human liver are being increasingly utilized to evaluate compound (drugs/chemicals) metabolism and toxicity. However, current in vitro models of the human liver are unable to determine the effects of compounds on the three major compartments of the liver, namely hepatic, vascular, and biliary, and how toxicity to one compartment may affect the other compartments. Similarly, while there has been some progress in developing implantable liver tissue surrogates as cell-based therapies for patients suffering from end-stage liver failure, such tissues do not contain the above-mentioned liver compartments with physiological interconnections. Our studies have shown that primary human hepatocytes (PHH) and liver endothelial cells (LEC) display high levels of in vivo-like functions for 4+ weeks in vitro when organized into 3-dimensional (3D) extracellular matrix (ECM) microgels that are generated using a high-throughput droplet microfluidics platform (so-called microtissues). This microtissue technology is uniquely suited to control the microenvironment of liver cells and could potentially protect cells from the shear stress induced via 3D bioprinting. Furthermore, we have shown that cholangiocytes display sprouting behavior in decellularized liver ECM (dECM) but not in collagen-I or Matrigel alone and such sprouting behavior can be directed via 3D bioprinting. In this high-risk/high-reward R21 proposal, we will leverage these platforms and findings to test the novel hypothesis that a 3D-printed biomaterial scaffold containing hepatic microtissues and liver dECM can be used to generate liver-like functional and integrated compartments (vascular, hepatic, and biliary). In aim 1, we will fabricate and characterize 3D- printed structures containing hepatic microtissues and LEC-lined vascular channels, while in aim 2, we will incorporate cholangiocytes into the biofabricated structures and investigate the ability to control and detect bile flow. If successful, our efforts will yield a first-of-its-kind scalable 3D-printed human liver tissue containing integrated hepatic, vascular, and biliary compartments that displays stable levels of diverse liver functions for several weeks in vitro. Ultimately, our 3D-printed human liver tissue can be used for investigating the effects of compounds on all three compartments of the liver and their interactions, as well as for implanting into animal models as potential cell-based therapy for chronic liver disease and acute liver failure.